State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, China.
Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of Marine Science, Hainan University, Haikou, China.
Front Immunol. 2023 Feb 14;14:1126843. doi: 10.3389/fimmu.2023.1126843. eCollection 2023.
Insulin-like growth factor binding protein 5 (IGFBP5) exerts an essential biological role in many processes, including apoptosis, cellular differentiation, growth, and immune responses. However, compared to mammalians, our knowledge of IGFBP5 in teleosts remains limited.
In this study, TroIGFBP5b, an IGFBP5 homologue from golden pompano () was identified. Quantitative real-time PCR (qRT-PCR) was used to check its mRNA expression level in healthy condition and after stimulation. overexpression and RNAi knockdown method were performed to evaluate the antibacterial profile. We constructed a mutant in which HBM was deleted to better understand the mechanism of its role in antibacterial immunity. Subcellular localization and nuclear translocation were verified by immunoblotting. Further, proliferation of head kidney lymphocytes (HKLs) and phagocytic activity of head kidney macrophages (HKMs) were detected through CCK-8 assay and flow cytometry. Immunofluorescence microscopy assay (IFA) and dual luciferase reporter (DLR) assay were used to evaluate the activity in nuclear factor-κB (NF-κβ) pathway.
The TroIGFBP5b mRNA expression level was upregulated after bacterial stimulation. , TroIGFBP5b overexpression significantly improved the antibacterial immunity of fish. In contrast, TroIGFBP5b knockdown significantly decreased this ability. Subcellular localization results showed that TroIGFBP5b and TroIGFBP5b-δHBM were both present in the cytoplasm of GPS cells. After stimulation, TroIGFBP5b-δHBM lost the ability to transfer from the cytoplasm to the nucleus. In addition, rTroIGFBP5b promoted the proliferation of HKLs and phagocytosis of HKMs, whereas rTroIGFBP5b-δHBM, suppressed these facilitation effects. Moreover, the antibacterial ability of TroIGFBP5b was suppressed and the effects of promoting expression of proinflammatory cytokines in immune tissues were nearly lost after HBM deletion. Furthermore, TroIGFBP5b induced NF-κβ promoter activity and promoted nuclear translocation of p65, while these effects were inhibited when the HBM was deleted.
Taken together, our results suggest that TroIGFBP5b plays an important role in golden pompano antibacterial immunity and activation of the NF-κβ signalling pathway, providing the first evidence that the HBM of TroIGFBP5b plays a critical role in these processes in teleosts.
胰岛素样生长因子结合蛋白 5（IGFBP5）在许多过程中发挥着重要的生物学作用，包括细胞凋亡、细胞分化、生长和免疫反应。然而，与哺乳动物相比，我们对鱼类 IGFBP5 的了解仍然有限。
在这项研究中，从金鲳鱼中鉴定出 IGFBP5 同源物 TroIGFBP5b。使用定量实时 PCR（qRT-PCR）检测其在健康状态和刺激后的 mRNA 表达水平。进行过表达和 RNAi 敲低实验，以评估其抗菌特性。构建了一个缺失 HBM 的突变体，以更好地理解其在抗菌免疫中的作用机制。通过免疫印迹法验证了亚细胞定位和核转位。进一步通过 CCK-8 检测法和流式细胞术检测头肾淋巴细胞（HKL）的增殖和头肾巨噬细胞（HKM）的吞噬活性。免疫荧光显微镜检测（IFA）和双荧光素酶报告（DLR）检测用于评估核因子-κB（NF-κβ）通路的活性。
细菌刺激后 TroIGFBP5b mRNA 表达水平上调。过表达 TroIGFBP5b 显著提高了鱼类的抗菌免疫能力。相反，TroIGFBP5b 敲低显著降低了这种能力。亚细胞定位结果表明，TroIGFBP5b 和 TroIGFBP5b-δHBM 均存在于 GPS 细胞的细胞质中。刺激后，TroIGFBP5b-δHBM 丧失了从细胞质向细胞核转移的能力。此外，rTroIGFBP5b 促进了 HKL 的增殖和 HKM 的吞噬作用，而 rTroIGFBP5b-δHBM 则抑制了这些促进作用。此外，HBM 缺失后，TroIGFBP5b 的抗菌能力被抑制，其在免疫组织中促进促炎细胞因子表达的作用几乎丧失。此外，TroIGFBP5b 诱导 NF-κβ 启动子活性并促进 p65 的核转位，而当 HBM 缺失时，这些效应被抑制。
综上所述，我们的结果表明，TroIGFBP5b 在金鲳鱼的抗菌免疫和 NF-κβ 信号通路激活中发挥重要作用，首次证明 TroIGFBP5b 的 HBM 在这些过程中在鱼类中起着关键作用。
